The popliteal lymph node assay: results of a preliminary interlaboratory validation study.

The popliteal lymph node (PLN) assay was proposed to predict possible autoimmune effects of xenobiotics. A preliminary interlaboratory validation study of the PLN assay was conducted in Wistar rats. Three laboratories tested in blind fashion four compounds, namely chlorpromazine, zimeldine, hydrazine and streptozotocin, which were reported to cause autoimmune-like reactions in humans, and one compound, i.e. barbital, which was not, using strictly the same experimental procedure. All tested substances were injected into the hind footpad of rats on day 1, and PLN weight and cellularity were measured on day 8. Comparison of the controlateral PLN was used to calculate weight and cellularity indices. The results were independently analyzed in a fourth laboratory. All four positive compounds were detected by the three laboratories using both weight and cellularity indices, and the negative compound consistently proved negative. Despite variations in absolute values between laboratories, although not significant, these results provide further evidence of the potential predictive value of the PLN assay.

Observation of hepatotoxic effects of 2-n-pentylaminoacetamide (Milacemide) in rat liver by a combined in vivo/in vitro approach.

Milacemide or 2-n-pentylaminoacetamide hydrochloride, a new glycine derivative, was found to cause elevations of plasma transaminases in patients suffering from severe depression and Alzheimer's disease. However, no signs of liver toxicity were observed during the course of earlier conducted subchronic and chronic in vivo studies in rodents and cynomolgus monkeys. In this study an in vivo/in vitro approach has been proposed to detect early alterations in key metabolic and functional liver capacities. Milacemide was administered by continuous i.v. infusion for 7 days to male Sprague-Dawley rats using subcutaneously implanted osmotic pumps. Doses were given of 0, 250 and 500 mg/kg per day. Body weight and food intake were recorded and at day 7 of exposure, Milacemide concentration, glucose, urea, triglycerides and cholesterol levels and alanine (ALT) and aspartate aminotransferase (AST) activities were measured in plasma. Non-esterified fatty acids were determined in serum. On day 8, after overnight fasting, hepatocytes were isolated. A portion of the cells derived from untreated animals (no osmotic pumps) were cultured in a primary monolayer and exposed in vitro to different Milacemide concentrations. The xenobiotic biotransformation capacity of the isolated hepatocytes was studied by measuring the cytochrome P450 content, ethoxycoumarin-O-deethylase (ECOD), pentoxyresorufin-O-deethylase (PROD), ethoxyresorufin-O-deethylase (EROD), aldrin epoxidase (AE), epoxide hydrolase (EH) and glutathione S-transferase (GST) enzyme activities. Triglycerides, cholesterol and phospholipid contents were measured on the isolated cells. At plasma concentrations of 43 and 130 microM Milacemide, the ALT activity was unchanged or significantly decreased, whereas the AST activity was increased in both cases. Other clinical chemistry parameters remained unchanged. Weight gain was significantly lower in rats treated with the high Milacemide dose. In addition, decreased food consumption was observed in all treated animals leading to significantly lower food efficiency factors for the rats treated with the high dose. Milacemide had a specific inhibitory effect on xenobiotic biotransformation: ECOD activity decreased to 60% of the control value for both Milacemide doses, PROD activity remained unaffected whereas EROD activity decreased to 65% of the control value. A decrease was also observed at the highest drug concentration for AE (to 41%), EH (to 65%), cytochrome P450 content (to 80%) and GST (to 85%). At 500 mg Milacemide kg/day, hepatocyte triglycerides levels increased 3.1-fold while cholesterol and phospholipid levels remained unaffected. Electron and light microscopy on total liver and isolated hepatocytes indicated a concentration-dependent accumulation of lipid droplets, the occurrence of numerous vacuoles in the cytoplasm and other structural abnormalities. When the cultured hepatocytes of control animals (without osmotic pumps) were exposed to Milacemide, the appearance of vacuoles and myeloid bodies could be confirmed in vitro. The results of this study using an in vivo/in vitro approach clearly show potential hepatotoxic properties of Milacemide, an effect not observed in conventional toxicity studies.

Fasting for 24 h reveals liver microsteatosis after continuous i.v. infusion of milacemide in the rat.

Milacemide (2-n-pentylaminoacetamide) hydrochloride was administered by continuous i.v. infusion for up to 7 days, at 300 and 600 mg/kg per day to male Sprague-Dawley rats. This was intended to provide high and sustained exposure to evaluate the effect of a preterminal 24-h fast on liver lipid content. Liver lipid content, as assessed by triglyceride concentration and histopathology, was not different in saline controls or rats infused with up to 600 mg/kg per day for up to 7 days, when they had access to food up to sacrifice. When the rats were fasted for 24 h before sacrifice, milacemide produced microsteatosis in the periportal and midzonal areas. The effect was significant after 2 days of infusion at 600 mg/kg per day and increased in intensity with duration of administration. After 7 days of infusion, at 600 mg/kg per day, liver triglycerides increased by more than 4-fold in rats fasted for the last 24 h. No other differences from the controls were observed at light microscopy or in liver protein content and AST activity. Liver ALT activity was decreased by 28% and plasma ALT activity by 23%. Plasma triglyceride levels were lowered by milacemide, in both fasted and fed rats. This study demonstrates that fasting for 24 h triggers the development of liver microsteatosis in rats exposed to milacemide. Fasting has been previously described to increase liver microsteatosis after administration of sodium valproate, 4-en valproate and pentenoic acid in the rat. These findings might help to identify the mechanism of the hepatic effects of milacemide.(ABSTRACT TRUNCATED AT 250 WORDS)

Isozyme characterization of cattle (Bos taurus) and American buffalo (Bison bison) cell cultures.

Four Bovidae cell lines (BEK-1, MDBK, Bu and EBTr) were characterized by means of enzymatic biochemical markers. Out of 15 enzymatic systems, 3--adenosine deaminase (Ada), phosphoglucomutase (Pgm) and nucleoside phosphorylase (Np)--were found to be polymorphic and quite suitable for biochemical identification of each cell line. The Bu cell line has shown a Np phenotypic pattern which could be distinctive of the Bison bison species.